An Image Cytometer is requested, because in addition to fluorescence and laser scatter signals, these platforms allows for discrimination and analysis of cell types and staining patterns not feasible by conventional flow cytometry. Two instruments were considered, the Compucyte iCys and the Amnis ImageStream 100. Based on the needs of Roswell investigators, the Amnis Image cytometer was selected. The ImageStream platform is designed to acquire six multispectral images of each cell (four fluorescent and two scatter parameters) at rates of 5,000 cells per minute. Cells are analyzed in suspension by hydrodynamically focusing them into a single-file core stream which passes through an Argon 488 nm excitation line. Light collected from the cells is ultimately projected onto a custom charge-coupled detector (CCD). This image processing occurs in real time during the image formation process and is saved to file. The ImageStream includes a sophisticated software platform which allows for the visualization and photometric/morphometric analysis of data files containing imagery from all cells analyzed, thereby combining quantitative image analysis with the statistical power of flow cytometry. Gates can be drawn on the plots to define sub-populations which can then be inspected in the gallery. The software also allows the user to create and combine parameters from any number of images of the same cell. For example, a nuclear image mask can be subtracted from the brightfield image of the entire cell to generate a mask that includes only the cytoplasmic region. There are a number of research questions that investigators at Roswell Park Cancer Institute (RPCI) and at SUNY Buffalo wish to ask which require an Image Cytometer; some of these are detailed in this application. These generally focus on using the instrument to (i) better analyze apoptosis, (ii) measure co-localization of fluorescent probes and translocation of molecules from the cytoplasm to the nucleus, and (iii) resolving fluorescent staining patterns on cells. One project will evaluate the potentiating effects of mild thermal exposure on T lymphocytes with the objective of using it to activate anti-tumor immune responses and potentiate vaccination strategies. Two projects are interested in quantifying the translocation of the cellular protein NF-?B from the cytosol to the nucleus. One will test the hypothesis that cellular distribution of NF-?B can be used as a determinant of response in AML patients treated with a new generation of targeted therapies. While many of these measurements can be performed by fluorescent or confocal microscopy, their laborious nature limits their applicability to qualitative assessments of small populations of cells and is potentially subject to operator bias. An Image Cytometer system would bring new quantitative capabilities to our traditional flow cytometry systems, combining quantitative image analysis with the statistical power of flow cytometry. Microscopists using the system will be able to analyze significantly larger cell populations easily and quickly and flow cytometrists will have access to detailed images of every cell analyzed. The instrument requested is a next generation cytometer which combine the efficiency and quantitative nature of flow cytometry with the visual power of microscopy. All the projects described in this application are relevant to the treatment of cancer and obtaining a better understanding of its biology. [unreadable] [unreadable] [unreadable]